The adjacent flanking region plays a critical role in facilitating the presentation of the Listeria monocytogenes product lemA to H2 M3(wt)-restricted, peptide-specific murine CD8 cells

Mice infected with Listeria monocytogenes (LM) generate CD8 effecters speci fic for f-MIGWII, the amino terminus of the bacterial product lemA presente d by the class Ib MHC molecule H2 M3(wt). lemA has several distinctive prop erties: 1) it is readily presented as an exogenous Ag in the absence of bac terial infection; 2) it is processed by a TAP-independent pathway, which is sensitive to chloroquine, pepstatin, and brefeldin; and 3) the immunogenic portion of the molecule is extremely resistant to proteolytic degradation even by proteinase K, To assess the structural basis for these findings, we expressed a truncated variant (t-lemA) containing the amino-terminal hexap eptide and the subsequent 27 amino acids linked to a histidine tail in Esch erichia coli, and purified the product by affinity chromatography. Purified t-lemA could be presented to f-MIGWII-specific effecters by macrophages an d fibroblasts at 1-10 nM. Unlike f-MIGWII, which binds directly to H2 M3(wt ), t-lemA required processing by a chloroquine-, pepstatin-, and brefeldin- sensitive pathway, Brefeldin sensitivity often implies endogenous processin g in the cytoplasm, but several lines of evidence suggest translocation to the cytoplasm and proteosomal degradation are not critical for t-lemA prese ntation. Unlike f-MIGWII, t-lemA was profoundly resistant to proteinase K, and, using S-35-labeled t-lemA, we could identify the region from position 1 to similar to 30 as the protease-resistant element. Thus, the hydrophobic peptide sequence following f-MIGMII can account for the unusual properties of lemA noted above. Analogous modification could be used to alter the pro perties of other peptide Ags presented by class I MHC products.